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Hierarchical control versus autoregulation of carbohydrate utilization in bacteriaGunnewijk, M.G W; van den Bogaard, P.T.C.; Veenhoff, Liesbeth M.; Heuberger, E.H M L; deVos, W.M.; Kleerebezem, M.; Kuipers, Oscar; Poolman, BerendPublished in:Journal of Molecular Microbiology and Biotechnology

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Regulation of Carbohydrate Utilization 401

Hierarchical Control versus Autoregulation of

Carbohydrate Utilization in Bacteria

*For correspondence. Email B.Poolman@chem.rug.nl;Tel. (+3150) 3634190; Fax. (+3150) 3634165.

J. Mol. Microbiol. Biotechnol. (2001) 3(3): 401-413.

© 2001 Horizon Scientific Press

JMMB Symposium

M. G. W. Gunnewijk1, P. T. C. van den Bogaard2,

L.M. Veenhoff1, E.H. Heuberger1, W. M. de Vos2,

M. Kleerebezem2, O. P. Kuipers2,3 and B. Poolman1*

1Department of Biochemistry, Groningen BiomolecularSciences and Biotechnology Institute, University ofGroningen, Nijenborgh 4, 9747 AG Groningen,The Netherlands2Wageningen Centre for Food Sciences, NIZO foodresearch, Department of Flavour and NaturalIngredients, P.O. Box 20, 6710 BA Ede, The Netherlands3Present address: Department of Genetics, GroningenBiomolecular Sciences and Biotechnology Institute,University of Groningen, Kerklaan 30, 9751 NN Haren,The Netherlands

Abstract

The involvement of phosphoenolpyruvate:sugar

phosphotransferase (PTS) proteins, like HPr and IIAGlc,

in the regulation of carbohydrate utilization has been

well established in Gram-negative and Gram-positive

bacteria. The majority of the studies of PTS-mediated

regulation have been concerned with the hierarchical

control of carbohydrate utilization, which results in the

preferential utilization of a particular carbohydrate from

a mixture of substrates. The underlying mechanisms

of PTS-mediated hierarchical control involve the

inhibition of expression of other catabolic enzymes

and transporters and/or the allosteric regulation of

their activity, which prevents the transcriptional

inducer to be formed or taken up into the cell. More

recently, it has become clear that PTS components

allow also the cell to tune the uptake rate(s) to the

carbohydrate availability in the medium and the

metabolic capacity of the cell. The different

phosphorylated species of HPr play a central role in

this autoregulatory control circuit, both at the gene

and at the protein level. Our knowledge of hierarchical

control and autoregulation of carbohydrate utilization

in bacteria is discussed.

Introduction

The first step in the metabolism of almost any carbohydrateis the transport of the molecule into the cell. In bacteriacarbohydrates are taken up by primary or secondarytransport systems or group translocation systems. Primarytransport of sugars is driven by ATP (or related energy-rich compounds), whereas secondary transport is drivenby the electrochemical gradient(s) of the translocated

solute(s) across the membrane (Poolman and Konings,1993). Among the secondary transport systems one candistinguish symporters (cotransport of two or more solutes),uniporters (transport of one molecule) and antiporters(countertransport of two or more solutes). Sugarsymporters usually couple the uphill movement of the sugarto the downhill movement of a proton (or sodium ion), i.e.,the electrochemical proton (or sodium ion) gradient drivesthe accumulation of sugar. Sugar uptake by grouptranslocation is unique for bacteria and involvesphosphoenolpyruvate:sugar phosphotransferase systems(PTSs) (Postma et al., 1993). The PTS catalyzes the uptakeof sugar concomitant with its phosphorylation. Thephosphoryl group is transferred from phosphoenolpyruvate(PEP) via the general energy coupling proteins Enzyme Iand HPr, and the sugar-specific phosphoryl transferproteins/domains IIA and IIB. IIB~P transfers thephosphoryl group to the sugar that is translocated via thesugar-specific IIC protein/domain (Figure 1). IIA, IIB andIIC can be separate proteins, domains in a singlepolypeptide or linked as pairs in any possible combination(Robillard and Lolkema, 1988; Saier and Reizer, 1992;Lengeler et al., 1994).

Most bacterial cells have the capacity to utilize severalcarbohydrates as carbon and energy source and possessvarious transport proteins and catabolic enzymes for themetabolism of the different carbohydrates. In addition,different mechanisms that control the transport and the firststeps of metabolism of a particular carbohydrate haveevolved. These mechanisms generally result in a sequentialuptake and metabolism of the available carbohydrates intime and/or a tuning of the metabolic rate to the needs ofthe cell. These two regulatory phenomena, hereafterreferred to as hierarchical control and autoregulation, areuniversal and have been reported for many bacteria.Hierarchical control of carbohydrates has been explainedby (i) inhibition of expression of genes encoding enzymesthat are involved in transport and metabolism of lesspreferred carbohydrates (Cohn and Horibata, 1959); (ii)inhibition of activity of enzymes that effect the uptake orproduction of the transcriptional inducer (hereafter referredto as inducer exclusion; Mcginnis and Paigen, 1969; Dillset al., 1980); and (iii) stimulation of efflux of intracellularinducer, that is, the carbohydrate or the phosphorylatedderivative (this phenomenon is referred to as inducerexpulsion, Reizer and Panos, 1980; Thompson and Saier,1981; Romano et al., 1987). The inducer exclusion andexpulsion mechanisms result in a lowering of theintracellular inducer concentration, and, thereby, indirectlyaffect gene expression. Autoregulatory control ofcarbohydrate utilization, on the other hand, occurs viaadjustment of the rate of transport of a particularcarbohydrate to the rate of its metabolism and theavailability of the substrate, hereby providing a feedbackor feedforward control to the pathway. Like with hierarchicalcontrol, autoregulation involves both control of gene

402 Gunnewijk et al.

transcription and control of enzyme/transporter activity. Thetwo regulatory mechanisms differ in the sense thatautoregulation of carbohydrate utilization controls thecatabolic activities within a specific metabolic pathway,whereas hierarchical control involves the metabolicpathway of the preferred carbohydrate as well as that ofthe less preferred carbohydrate.

The mechanisms that result in the regulation of theinitial steps of carbohydrate metabolism have been studiedin two groups of bacteria: Gram-negative, enteric bacteria,such as Escherichia coli and Salmonella typhimurium; andGram-positive, low GC bacteria, such as Bacillus subtilisand several streptococcal and lactobacilli species. Forthese groups of bacteria it has been well-established thatthe PTS system plays a crucial role in the mechanismsunderlying hierarchical control of carbohydrate utilization.More recent experiments have indicated that autoregulationof transport and metabolism of carbohydrates is alsocontrolled by the PTS.

How does the PTS exert all these regulatory functions?The PTS is able to sense the availability of carbohydratesand the metabolic capacity of the cell to metabolize thesecarbohydrates by adjusting the phosphorylation state ofthe PTS components; key players are the IIAGlc and HPrproteins. As the phosphoryltransfer steps in the PTS systemare reversible, the addition of a PTS substrate to the cellinduces a dephosphorylating signal that is transmittedeither directly, via IICBGlc in the case of glucose, to thecentral regulatory proteins IIAGlc and HPr or indirectly by

rerouting the phosphoryl transfer to other substrate-specificEII complexes, resulting in reduced ratios of HPr(His~P)/HPr and IIAGlc~P/IIAGlc (Figure 1 and 2; Postma et al.,1993). On the contrary, in the absence of a PTS substrate,a high [PEP]/[pyruvate] ratio will favor the histidine-phosphorylated state of HPr and IIAGlc (Weigel et al., 1982a;Hogema et al., 1998b). Besides being phosphorylated atHis-15, the HPr protein of several Gram-positive bacteriacan also be phosphorylated at Ser-46 in an ATP-dependentprotein kinase catalyzed reaction (Deutscher and Saier,1983; Reizer et al., 1984). The reverse reaction, thehydrolysis of HPr(Ser-P), is catalyzed by a cytosolicHPr(Ser-P)phosphatase, which is stimulated by highconcentrations of phosphate (Deutscher et al., 1985).HPr(Ser)kinases of several Gram-positive bacteria,including B. subtilis, Streptococcus pyogenes, Lactobacillusbrevis and Lactococcus casei, are stimulated by earlyglycolytic intermediates, in particular fructose 1,6-bisphosphate (FDP) (Deutscher and Engelman 1984;Reizer et al., 1984; 1988; 1998; Galinier et al., 1998 andDossonnet et al., 2000). The HPr kinases from S. salivarius,S. mutans Ingbritt and E. faecalis do not seem to bestimulated by FDP or other glycolytic intermediates, butinstead, these enzymes are controlled by ATP and Pi levelsin the cell (Thevenot et al., 1995; Brochu et al., 1999,Kravanja et al., 1999). Recent experiments on theHPr(Ser)kinase of B. subtilis showed that stimulation byFDP essentially occurred at low ATP and enzymeconcentrations, and that positive cooperativity for FDP

Figure 1. Schematic representation of the phosphoenolpyruvate:glucose phosphotransferase system of Gram-negative enteric bacteria. The central role ofIIAGlc(~P) in controlling transcription and inducer exclusion is illustrated. EI, Enzyme I; Glc-6P, glucose-6P; CRP, cAMP receptor protein; RNA polym., RNApolymerase; R, repressor; C+, cation; S, secondary transport protein; GK, glycerol kinase; and GlpF, glycerol facilitator.

IIA IIBIIC

GK

Glp

F

S

IIA

IIA~P

IIB IIC

AC

R

RNA polym. CRP

Regulation of Carbohydrate Utilization 403

binding is related to oligomerization of the enzyme (Jaultet al., 2000). Overall, the formation of HPr(Ser-P) isproposed to be governed by the relative cellularconcentrations of ATP, Pi and/or FDP, which are indicatorsof the energy status of the cells (Mason et al., 1981;Thompson and Torchia, 1984). This is supported by directobservations on the relative levels of HPr, HPr(Ser-P),HPr(His~P) and the doubly phosphorylated speciesHPr(Ser-P/His~P) in the cell. It has been shown that,HPr(Ser-P) is the dominant phosphorylated form of HPr inrapidly growing streptococcal cells, whereas free HPr andHPr(His~P) are the major species in slowly growing cells(Vadeboncoeur et al., 1991; Thevenot et al., 1995;Gunnewijk and Poolman, 2000a). HPr(Ser-P/His~P) isalways a minor species (ranging from 5 – 30 % of totalHPr present in different streptococci) and only present inrapidly growing cells; its physiological function has not beenstudied in detail.

This paper describes how the phosphorylated statesof the PTS proteins control the transport and metabolismof carbohydrates. Specifically, their role in controllinghierarchical utilization is discussed as well as the recentlydescribed role of the PTS in the autoregulation ofcarbohydrate utilization. The former focuses mainly on thedifferent mechanisms of hierarchical control that haveevolved in Gram-negative enteric and low GC Gram-positive bacteria. It becomes clear that in some speciesmore than one PTS-mediated regulatory mechanism isoperative. The autoregulatory mechanism follows primarilyfrom recent observations made for lactose transport andmetabolism in Streptococcus thermophilus. Thisautoregulatory mechanism is unique for its involvement ofa IIA-like protein, which is unusual for PTS-mediatedregulation in Gram-positive bacteria.

Hierarchical Control of Carbohydrate Utilization

Hierarchical control of carbohydrate utilization was firstdescribed by Monod in 1942. This study demonstrated thatwhen E. coli was grown on a mixture of carbohydrates, itsgrowth curve was biphasic as a result of the sequentialuse of carbohydrates. For instance, glucose is used firstwhen present in combination with lactose, melibiose,maltose and/or raffinose. The molecular basis forhierarchical carbohydrate utilization is well understood, inparticular in enteric bacteria, and is generally referred toas catabolite repression. Catabolite repression is definedas the inhibitory effect of a preferred carbohydrate on theexpression of other (catabolic) genes, and in fact, alsoincludes inducer exclusion and inducer expulsion. Theselatter two regulatory phenomena have in common that theintracellular inducer concentration will be reduced andthereby, indirectly, affect gene expression. Most often theactivity of the transport protein is modified such that theuptake of the inducer is prevented or the accumulatedinducer is expelled from the cell. In some cases the firststep(s) of the metabolism that produce(s) the transcriptionalinducer is inhibited. The different mechanisms of inducerexclusion and inducer expulsion will be discussed in detailbelow. In the control of gene expression not only “inducer-specific” transcription factors, but also “general” ones areinvolved. In enteric bacteria, general transcriptional controlis mediated by CRP (cAMP receptor protein), whichrequires cAMP as cofactor. The synthesis of cAMP iscatalyzed by adenylate cyclase, which is under PTS controlas it can be activated by IIAGlc~P. In Gram-positive bacteriacAMP is not present and CcpA, the equivalent of CRP, isregulated by HPr(Ser-P). In addition to these strategiesfor controlled gene expression, Gram-negative and Gram-

Figure 2. Schematic representation of the phosphoenolpyruvate:glucose phosphotransferase system of Gram-positive bacteria. The central role of the HPrspecies in controlling transcription, inducer exclusion, inducer expulsion and inducer control is illustrated. CRE, Catabolite Responsive Element; FDP,fructose-1,6-bisphosphate; and Pi, free phosphate. Other symbols as in the legends of Figure 1.

CcpA RNA polym.

HPr(Ser-P)

HPr

HPr(Ser-P)

HPr(His~P)

Pasell

S

S

IIC

IIA

IIB

IIC

IIB IIC

GK

Gpl

F

404 Gunnewijk et al.

positive bacteria have also developed transcriptionalregulators that contain PTS regulation domains (PRD-containing proteins). In the next four sections the abovementioned PTS-mediated mechanisms underlyingcatabolite repression are discussed.

Catabolite Repression by Inducer Exclusion or Inducer

Expulsion

Inducer exclusion is established by different mechanismsin Gram-negative and Gram-positive bacteria and involvesdifferent PTS proteins. In Gram-negative enteric bacteriainducer exclusion is determined by the phosphorylationstate of IIAGlc (Figure 1). In the presence of glucose, theactivities of non-PTS carbohydrate transport proteinsspecific for lactose, maltose, melibiose or raffinose areinhibited by allosteric interaction of the unphosphorylatedform of IIAGlc (Nelson et al., 1983; Postma et al., 1984;Misko et al., 1987; Dean et al., 1990; Titgemeyer et al.,1994; Hoischen et al., 1996). Not only transport proteins,but also catabolic enzymes can be inhibited via allostericinteraction with IIAGlc, as observed for the glycerol kinase(Postma et al., 1984; Novotny et al., 1985). Inhibition ofglycerol kinase prevents the formation of L-glycerol-3-phosphate, the inducer for transcription of the glp operon,and thus prevents the expression of the catabolic enzymesspecific for glycerol metabolism. This example of inducerexclusion is well-understood at a molecular level as thecomplex of glycerol kinase and IIAGlc has been crystallizedand the structure has been elucidated at 2.6 Å resolution(Hurley et al., 1993). These studies have revealed that E.coli IIAGlc binds to E. coli glycerol kinase at a region that isdistant from the catalytic site of glycerol kinase, suggestingthat long-range conformational changes mediate theinhibition of glycerol kinase by IIAGlc. The binding of glycerolkinase to IIAGlc involves mainly hydrophobic andelectrostatic interactions, but also one hydrogen bond,involving an uncharged aspartate and a Zn(II) binding site(Feese et al.,1994). The Zn(II) binding site is made up ofthe two active-site histidines of IIAGlc (His75 and His90),Glu478 of glycerol kinase and a H2O molecule.Phosphorylation of IIAGlc destroys the intermolecular Zn(II)binding site and disrupts the interactions between IIAGlc

and glycerol kinase.In Gram-positive bacteria, inducer exclusion does not

involve regulation by IIAGlc, but rather allosteric control byHPr(Ser-P) or control via HPr(His~P)-dependentphosphorylation (Figure 2). In L. brevis, HPr(Ser-P) hasbeen implicated in the control of uptake of non-PTScarbohydrates such as glucose, lactose and ribose (Ye etal., 1994 a,b). Recent studies in ptsH and hprK mutants ofL. casei showed that HPr(Ser-P) can also act in inducerexclusion by inhibiting the uptake of the non-PTScarbohydrate maltose (Viana et al., 2000; Dossonet et al.,2000). In some Gram-positive bacteria, like E. faecalis, E.casseliflavus and B. subtilis, the concentration of thetranscriptional inducer for the glp-operon is controlled viathe activity of glycerol kinase. The net result of thisregulation is equivalent to the allosteric control of glycerolkinase from enteric bacteria by IIAGlc, but the underlyingmechanisms are different. In the Gram-positive bacteria,glycerol kinase is stimulated via HPr(His~P)-dependentphosphorylation (Deutscher and Sauerwald 1986; Wehtje

et al., 1995; Charrier et al., 1997). The phosphorylation ofglycerol kinase by HPr is reversible, and thedephosphorylated, less active form of glycerol kinase isdominant when a PTS-substrate is present in the medium(Deutscher et al., 1993). HPr also affects the hierarchicalutilization of PTS carbohydrates via competition forHPr(His~P), a general mechanism that is operative in bothGram-negative and Gram-positive bacteria. As the affinityof HPr(His~P) varies for the carbohydrate-specific IIAproteins/domains, competition for HPr(His~P) leads tohierarchical uptake of PTS carbohydrates.

For some low-GC Gram-positive bacteria cataboliterepression is achieved by a mechanism in which the induceris expelled from the cell. Two types of inducer expulsionmechanisms have been demonstrated (Figure 2). Inhomofermentative lactic acid bacteria like E. faecalis, S.pyogenes, S. bovis and L. lactis, which transport mostsugars via PTS systems, lactose and glucose accumulatein the cytoplasm in their phosphorylated forms (Reizer andPanos, 1980; Thompson and Saier, 1981, Ye et al., 1996).Addition of a rapidly metabolizable sugar results indephosphorylation of the accumulated sugar-P, which isfollowed by a rapid efflux of the free sugar from the cell(Reizer et al., 1983). A sugar-P phosphatase (Pase II) hasbeen identified in all these four species and this enzymeseems to be absent in S. aureus, S. mutans, S. salivariusor B. subtilis, organisms that do not exhibit the sugar-Phydrolysis dependent expulsion phenomenon (Cook et al.,1995 a, b; Ye et al., 1996). Based on in vitro studies withtoluenized vesicles or purified Pase II, it has suggestedthat HPr(Ser-P) stimulates PaseII (Ye et al., 1994 c, d; Yeand Saier, 1995a). A second type of inducer expulsion wasobserved in heterofermentative lactobacilli such as L.brevis. These bacteria transport lactose and glucose viaproton symport mechanisms and accumulate thesesubstrates as free (non-phosphorylated) cytoplasmicsugars. Binding of HPr(Ser-P) to the glucose/H+ andlactose/H+ symporters is thought to alter their energycoupling mechanism, resulting in a conversion of thesystems from carbohydrate-proton symport intocarbohydrate uniport (Ye and Saier, 1995 a,b).Consequently, accumulated substrates are expelled fromthe cell, down the concentration gradient, and thereby theinducer levels are lowered (Romano et al., 1987; Ye et al.,1994 a, b).

Catabolite Repression by Regulation of cAMP

Synthesis

Catabolite repression in enteric bacteria involves inhibitionof the uptake of the inducer (inducer exclusion [Inada etal., 1996a; Kimata et al., 1997]) as well as a lowering ofthe cAMP levels via a diminished adenylate cyclase activity(Figure 1). In the absence of a PTS-carbohydrate, thephosphorylated form of IIAGlc activates adenylate cyclaseand stimulates cAMP production. Binding of cAMP to thecAMP receptor protein (CRP) enables the complex to bindto specific sites in several promoter regions and allowstranscriptional regulation (either positively or negatively)of at least 45 genes and operons in E. coli and S.typhimurium. This subject has been the topic of manyreviews and is not described further here (Botsford andHarman, 1992; Kolb et al., 1993; Postma et al., 1993).

Regulation of Carbohydrate Utilization 405

CcpA-Mediated Catabolite Repression

In many Gram-positive bacteria the general transcriptionfactor, CcpA, mediates the repression of several catabolicgenes (Figure 2) (Hueck and Hillen, 1995). HPr(Ser-P) hasbeen shown to interact with CcpA, allowing the latter proteinto bind specifically to a cis-acting sequence. This sequence,named catabolite-responsive element (cre), is present inor near the promoter regions of many catabolite repressionsensitive operons (Weickert and Chambliss, 1990; Fujitaet al., 1995; Deutscher et al., 1995; Henkin, 1996;Gösseringer et al., 1997; Kim et al., 1995). The HPr(Ser-P)/CcpA complex forms a ternary complex with cre,consisting of two molecules of HPr(Ser-P), the CcpA dimerand the cre sequence (Jones et al., 1997). Both theformation of the HPr(Ser-P)/CcpA-complex and its bindingto the cre sequences is stimulated by FDP (Deutscher etal., 1995; Kim et al., 1998). The histidine residue at position15 in the HPr protein, the active site for PEP-dependentEnzyme I phosphorylation, is important for CcpA-mediatedrepression, as mutation or phosphorylation of His-15 blocksthe interaction of HPr(Ser-P) with CcpA. This suggests alink between catabolite repression and PTS-mediatedcarbohydrate transport (Deutscher et al., 1995; Reizer etal., 1996). Indeed, the uptake of glucose or other rapidlymetabolizable PTS carbohydrates leads todephosphorylation of the PTS proteins and to an increaseof the concentrations of glycolytic intermediates thatactivate the HPr(Ser)kinase. As a result the levels ofHPr(Ser-P) rise and strong CcpA-dependent repression isobserved under these conditions, leading to hierarchicalcarbohydrate utilization.

Catabolite control by CcpA not only involves repressionbut also activation of genes and operons. In B. subtilis,transcription of the alsS and ackA genes, encoding α-acetolactate synthase and acetate kinase, respectively, isactivated by CcpA when glucose is present in the medium(Grundy et al., 1993; Renna et al., 1993). More directevidence for a link between catabolite control and glycolyticactivity has been reported for Lactococcus lactis. CcpAwas found to be a transcriptional activator of the las operon,

thereby controlling the production of the three key glycolyticenzymes, that are phosphofructokinase, pyruvate kinaseand lactate dehydrogenase (Luesink et al., 1998).

Catabolite Repression Involving PRD-Containing

Proteins

The expression of genes encoding enzymes of severalPTS-dependent metabolic pathways, such as the β-glucoside operons in E. coli and B. subtilis, the sucroseregulon and the levanase operon in B. subtilis, and themannitol operon in Bacillus stearothermophilus, arecontrolled by the PTS (Mahadevan and Wright, 1987; Crutzet al., 1990; Martin-Verstraete et al., 1990; Arnaud et al.,1992; Le Coq et al., 1995; Tobisch et al., 1999a; Stulke etal., 1997; Henstra et al., 1999). The transcription of theseoperons is initiated by specific activators, like LevR andMtlR, or controlled by specific antiterminators, like BglG,LicT and SacT. The activities of these transcriptionalregulators are controlled via the phosphorylation states oftheir PRD-domains (PTS Regulation Domain; reviewed byStulke et al., 1998). In the absence of PTS substrates, thephosphorylated IIB component of the carbohydrate-specificpermease phosphorylates a PRD, thereby inactivating theregulator and thus preventing the transcription of therespective operons (Figure 3A). In the presence of the PTSsubstrate, the phosphate group is transferred to the PTSsubstrate, leaving the regulator unphosphorylated andactive for induction of the operon (Figure 3B). In this waytranscription is controlled in response to the availability ofthe inducer. In addition, some of these PRD-containingtranscriptional regulators also require HPr(His~P)-dependent phosphorylation for activity (Stulke et al., 1995;Tobisch et al., 1999a; Görke and Rak, 1999; Henstra etal., 1999). In MtlR of B. stearothermophilus a IIA-likedomain is present in addition to two PRDs, which resultsin an intricate interplay of multiple (de)phosphorylationreations, that is HPr- and IIB-mediated as well asinterdomain phosphoryl transfer (Henstra et al., 2000).

Regulation of these transcription factors by thecarbohydrate-specific IIB and the general PTS protein HPr

Figure 3. Model for activity control of PRD-containing transcriptional regulators as suggested for BglG by Görke and Rak, 1999. (A) In the absence of anyPTS sugar the phosphorylated form of IIB phosphorylates the transcriptional regulator (R) leading to its monomer formation, the inactive form. In addition,HPr may phosphorylate R at a second site, which is without effect when R is phosphorylated by IIB. (B) In the presence of ß-glucosides (but absence of otherPTS substrates), IIB dephosphorylates R, which is necessary but not sufficient for its activation. HPr directly transfers phosphoryl groups to a distinct sitewithin R, which allows the protein to dimerize to the active form and stimulate transcription of the specific operon. (C) Appearance of any other PTS sugarleads to additional competition for phosphoryl groups and the transport of other PTS sugars reduces the level of HPr(His~P). As a consequence, HPr isunderphosphorylated and therefore unable to activate R by phosphorylation.

IIC

IIAIIBPP

IIC

IIAIIBPP

IIC

IIAIIBPP

IIC

IIAIIBPP

406 Gunnewijk et al.

is an example of an elegant dual control mechanism thatsenses both the presence of a specific PTS substrate andthe need of the cell to transport and metabolize thissubstrate. Since the level of HPr(His~P) is, amongst others,determined by the rate of uptake of the PTS carbohydrateitself, transcription is tuned (autoregulated) to the availabilityof the PTS substrate. The level of HPr(His~P) decreasesmore strongly with the presence of a more preferred PTScarbohydrates, due to a more rapid uptake or competitionof different IIA components for HPr(His~P), which leads tohierarchical carbohydrate utilization both at the level of PTSuptake and transcription. Besides this competition forHPr(His~P), the rapid metabolism of carbohydrates will leadto HPr(Ser-P) formation. This results in an additionaldecrease in the formation of HPr(His~P) as free HPr willbe no longer available for PEP-dependent phosphorylationby Enzyme I (Saier, 1989). Therefore, it has been proposedthat HPr(His~P)-dependent stimulation of transcriptionalregulators is another strategy by which cataboliterepression can be manifested in Gram-positive bacteria.Indeed, CcpA-independent catabolite repression isobserved for the bgl, lic and lev operons in ccpA mutantsof B. subtilis (Martin-Verstraete et al., 1995; Kruger et al.,1996; Tobisch et al., 1999b). This catabolite repression isdependent on the presence of a specific transcriptionalregulator (LicT, LicR or LevR).

Autoregulation of Carbohydrate Utilization

Besides hierarchical control, the PTS also mediatesautoregulation of carbohydrate utilization. The mechanisticconcepts of the autoregulatory control circuits are emergingand, in a few cases, it has been shown that the rate ofcarbohydrate uptake is tuned to the metabolic capacity ofthe cell and the carbohydrate availability in the medium. InGram-positive bacteria, HPr(Ser-P), which is formed at highrates of metabolism, is thought to autoregulate the uptakerate of PTS carbohydrates. In vitro experiments withmembrane vesicles of L. lactis demonstrated that HPr(Ser-P) inhibited PTS-mediated uptake of 2-deoxyglucose (2DG)and thiomethyl-β-galactoside (TMG) (Ye et al., 1994 c, d).In vivo studies with ptsH mutants of B. subtilis and S. aureusshowed that HPr(Ser) phosphorylation reduced thefermentation response of several PTS carbohydrates(Reizer et al., 1989a; Ye and Saier, 1996). Based on kineticstudies, it was suggested that HPr(Ser-P) inhibits PTSuptake by reducing the phosphoryltransfer rates fromEnzyme I via HPr to the IIA proteins (Reizer et al., 1989a,1992). When HPr was phosphorylated on Ser-46, theaffinity of Enzyme I and the affinities of several IIA proteinsfor HPr are reduced, and also the maximal velocities ofthese phosphoryl transfer reactions are reduced. It is worthnoting that this HPr(Ser-P)-mediated inhibition of PTScarbohydrate uptake does not create a hierarchy incarbohydrate utilization as the inhibition was general in allPTS systems tested.

In contrast to what might be expected, also themetabolism of non-PTS carbohydrates can trigger PTS-mediated mechanisms to autoregulate the rate ofmetabolism. In Gram-negative bacteria, non-PTScarbohydrates like glucose-6-P, gluconate or lactose inducecatabolite repression by decreasing the concentration of

both cAMP and CRP (Perlman et al., 1969; Okinaka andDobrogosz, 1966; Epstein et al., 1975; Inada et al., 1996b;Hogema et al., 1997). Moreover, there is evidence that themetabolism of non-PTS carbohydrates influences theintracellular [PEP]/[pyruvate] ratio (Hogema et al., 1998b).Since this ratio is the driving force for the phosphorylationof the PTS proteins, non-PTS carbohydrates can influencethe phosphorylation state of IIAGlc and thereby regulateIIAGlc-mediated inducer exclusion (as described earlier).In agreement are the characteristics of E. coli lacY mutantsthat express a lactose carrier protein, which is insensitivefor IIAGlc-mediated inducer exclusion. In this mutant lactoseis consumed much faster than in the wild type, largeramounts of glucose are excreted, and strongerdephosphorylation of IIAGlc is observed (Hogema et al.,1998b; 1999). The growth rate is retarded in strains in whichthe mutations leading to insensitivity to inducer exclusionare combined with an increased lac expression. On thebasis of these observations a model has been proposed,in which the metabolism of non-PTS carbohydratesdetermines the phosphorylation state of IIAGlc, and, therebycontrols inducer exclusion and transcriptional regulationvia control of adenylate cyclase activity (Hogema et al.1998b, 1999). This autoregulatory circuit is an importantmechanism for E. coli cells to adjust the uptake rate to itsmetabolic capacity.

Recently, in the Gram-positive organism S.thermophilus evidence was obtained for autoregulation ofthe transport of the non-PTS carbohydrate lactose. Thisinvolved the tuning of the uptake to the rate of sugarmetabolism. In the sections below we will discuss howtransport and metabolism of lactose is regulated at the levelof gene transcription and protein activity. The underlyingmechanisms involve the HPr(Ser-P)/CcpA complex andHPr(His~P)-mediated phosphorylation of the IIA-likedomain of the non-PTS transport protein, LacS.

Enzymatic and Structural Features of the LacS Protein

In S. thermophilus, lactose is taken up via the secondarytransport protein LacS (Poolman et al., 1996). LacScatalyzes two modes of transport, solute-H+ symport, drivenby the proton motive force (∆p) and lactose/galactoseexchange, which is driven by the concentration gradientsof lactose and galactose across the membrane (Foucaudand Poolman, 1992). The lactose/galactose exchangereaction via LacS is the most relevant transport mode invivo as it is much faster than the lactose/H+ symportreaction (Knol et al., 1996). In addition, the galactose moietyof lactose cannot be metabolized in most S. thermophilusstrains, and therefore galactose has to be expelled fromthe cell. Kinetic studies have revealed that the affinity ofLacS for galactose and lactose at the cytoplasmic bindingsite is 20-fold higher than at the extracellular binding site,and that in this conformation galactose is preferred overlactose (Veenhoff and Poolman, 1999). These observationsare consistent with the view that LacS is designed tocatalyze an efficient lactose/galactose exchange ratherthan a unidirectional lactose influx.

Although, the LacS protein is not a PTS transportsystem, the protein has a carboxyl-terminal hydrophilicdomain of about 160 amino acids, which is homologous toIIA proteins/domains of various PTS systems. Interestingly,

Regulation of Carbohydrate Utilization 407

the LacS protein is, amongst others, homologous to themelibiose transport proteins of S. typhimurium and E. coli,which lack a IIA-like domain, but are regulated by IIAGlc asdiscussed elsewhere (Poolman et al., 1996). Thehydrophilic domain of LacS, called IIALacS, has 34 % residueidentity with IIAGlc of E. coli and contains several residuesthat are conserved in the active site of PTS IIA proteins/domains (Poolman et al., 1989). These conserved residuesinclude His-552 and His-537, corresponding to His-90 andHis-75 of IIAGlc from E. coli, respectively. The formerhistidine residue has been shown to be the phosphoryl-accepting site for HPr(His~P) phosphorylation (Dörschuget al., 1984), whereas His-75 is involved in stabilization ofthe phosphoryl group, when bound to His-90, and in thetransfer of the phosphoryl group to the membrane proteinIICBGlc (Presper et al., 1989; Meadow and Roseman,1996).

On the basis of amino acid alignments, we anticipatedthat IIALacS would be able to carry out one or more functionsassociated with IIAGlc, i.e., phosphorylation by HPr(His~P),phosphoryl transfer to the glucose specific IIB domain and/or affecting other catabolic functions such as inducerexclusion of non-PTS substrates. To study the propertiesof the IIALacS domain, a IIALacS protein comprising thecarboxyl-terminal 173 residues of LacS was produced inE. coli (Gunnewijk et al., 1999). Biochemicalcharacterization of purified wild type and mutant IIALacS

protein showed that (i) HPr(His~P) can phosphorylateIIALacS; (ii) phosphorylation of IIALacS by HPr(His~P) fromS. thermophilus is 5 orders of magnitude slower thanphosphorylation of IIAGlc by HPr(His~P) from E. coli; (iii)the phosphorylation-site corresponds most probably to His-552; and (iv) IIALacS is unable to transfer the phosphorylgroup to E. coli IIBGlc. These results suggest that IIALacS

has evolved into a protein (domain), whose main functionis not to transfer phosphoryl groups rapidly. Instead, theunphosphorylated form of IIALacS was able to inhibit theactivity of glycerol kinase (inducer exclusion). The extentof inhibition of glycerol uptake was dependent on the actualuptake rate of glycerol [amount of glycerol kinase],suggesting that IIALacS inhibits glycerol kinase by forminga (stoichiometric) complex with the enzyme. In relation tothese regulatory properties of IIALacS, carbohydratefermentation studies in S. thermophilus showed that theIIALacS is involved in controlling the lactose uptake rate asdeletion of the IIALacS domain increases the rate of lactoseutilization relative to that of the wild type (Gunnewijk et al.,1997).

Regulation of Carbohydrate Utilization in

Streptococcus thermophilus

S. thermophilus has a very limited capacity to utilizecarbohydrates. Lactose and sucrose are fermented mostrapidly, glucose is used very slowly by most strains, andonly one or few other carbohydrates can be used by moststrains. S. thermophilus co-metabolizes sucrose andlactose, a PTS and a non-PTS substrate, respectively,indicating that the utilization of these carbohydrates is not(strongly) hierarchically controlled. Instead, it has beenproposed that HPr(His~P)-mediated regulation of thelactose uptake rate serves to control the flux of glycolysis.This mechanism is based on studies of the kinetic

properties of phosphorylated and unphosphorylated LacS,and on the LacS levels of the cell as a function of thephosphorylation state of HPr. The various species of HPrpresent in lactose-growing S. thermophilus cells have beenquantified at different stages of growth (Gunnewijk andPoolman, 2000). HPr(Ser-P) appears to be the dominantphosphorylated species in the exponentional phase ofgrowth, whereas HPr(His~P) dominates in the stationaryphase. Similar results were obtained when S. thermophiluscells were grown on sucrose (Gunnewijk and Poolman,unpublished results). The fact that the levels of HPr(Ser-P), HPr(His~P) and HPr are similar in sucrose- and lactose-growing cells suggest that the rate of glycolysis of bothcarbohydrates is sufficiently high to keep Ser-46phosphorylated and that the drain of phosphoryl transferto sucrose is minor compared to the phosphorylation activityof Enzyme I. The similar HPr(His~P)/HPr ratios alsosuggest that the PEP/pyruvate ratios are comparable insucrose and lactose growing cells. Although PEP levelshave not been measured in S. thermophilus, it has beenfirmly established for other lactic acid bacteria thatconcentrations of PEP are relatively low in rapidlymetabolizing cells, whereas PEP concentrations increaseunder conditions of carbohydrate limitation (Mason et al.,1981; Thompson and Torchia, 1984; Konings et al., 1989).The increase in HPr(His~P) in S. thermophilus at laterstages of growth would thus correlate with the increasedPEP levels and a decreased metabolic activity.

Regulation of the Lactose Transport System

The effect of HPr(His~P)-mediated phosphorylation onlactose transport has been studied in vitro using amembrane system, in which purified LacS protein wasincorporated into liposomes with the IIALacS domain facingoutwards (Gunnewijk and Poolman, 2000b). This systemallowed phosphorylation and manipulation of activity ofLacS by adding PEP, Enzyme I and/or HPr to the outsidemedium. Upon phosphorylation of LacS the maximal rateof lactose exchange transport was increased, whereas therate of ∆p-driven lactose uptake at low lactoseconcentrations was somewhat decreased. This apparentinhibition is in agreement with earlier results obtained withhybrid membrane vesicles, and reflects a somewhat loweraffinity of LacS for lactose when the protein is in thephosphorylated state. This inhibition is not observed whenthe activity of the protein is analyzed under saturatinglactose concentrations. Moreover, the inhibition of ∆p-drivenlactose uptake is not observed with LacS mutants that lackIIALacS [LacS(∆160)] or the active-site histidine residue[LacS(H552R)] (Poolman et al., 1995a). These resultsindicate that HPr(His~P)-mediated phosphorylation ofIIALacS at the conserved His-552 residue modulates lactosetransport activity of the LacS protein. In line with a rangeof kinetic studies (Foucaud and Poolman, 1992; Poolmanet al., 1995b), it has been proposed that phosphorylationaffects the rate constants for the reorientation of the ternarycomplex (LacS with bound lactose plus proton), which israte-determining for exchange transport but not for ∆p-driven uptake. Since the lactose/galactose exchangereaction and not the ∆p-driven uptake is most relevant inlactose (glycolysing)-metabolizing cells of S. thermophilus,HPr(His~P)-mediated phosphorylation of LacS evokes

408 Gunnewijk et al.

maximal activity of the lactose transport protein in vivo byincreasing the Vmax of the lactose/galactose exchangereaction. This condition is met in cells at the late-exponentional and stationary phase of growth, whenHPr(His~P) is the dominant species of HPr (Gunnewijk andPoolman, 2000a).

The transition from HPr(Ser-P) to HPr(His~P) at thelate-exponentional phase of growth parallels an increasein the extent of LacS phosphorylation, a decrease in lactoseand an increase in galactose concentration in the growthmedium. Since the Km

out for lactose is higher than that forgalactose (Veenhoff and Poolman, 1999), the decrease inlactose/galactose ratio in the medium will reduce thelactose transport capacity as growth proceeds. This will atsome point during growth be reflected in a reducedglycolytic activity, to which the HPr(Ser-P)/HPr(His~P) ratiois very sensitive (Reizer et al., 1984; 1989b; Deutscher etal., 1985; Deutscher and Engelman, 1984). By increasingthe specific activity upon HPr(His~P)-mediatedphosphorylation, S. thermophilus is able to compensatefor a decrease in lactose concentration (and galactoseaccumulation in the medium) by adjusting the lactoseuptake rate rapidly. Another, but slower, response involvesadjustment of the LacS expression levels, which isdescribed in the next section.

Transcriptional Control of the lac Operon in S.

thermophilus

The observed transition from HPr(Ser-P) to HPr(His~P) atthe late-exponential phase of growth parallels an increasein LacS level (Gunnewijk and Poolman, 2000). At stationaryphase, the expression level is about 10 times higher thanthe basal LacS level at early-exponential phase of growth,indicating that HPr(Ser-P) plays a role in repression of thelac operon. Consistent with a higher basal level of LacS inS. thermophilus ST11∆lacS/pGKhis, the increase inexpression was less than two-fold when early-exponentialand late-exponential or stationary phase were compared.Importantly, the time point of transition from HPr(Ser-P) toHPr(His~P) is dependent on the basal level of LacSexpression, as the transition occurred at later steps ofgrowth in S. thermophilus ST11∆lacS/pGKhis whencompared to the wild-type ST11 strain. These resultssuggest that the formation of HPr(Ser-P) is determined bythe rate of lactose transport and/or metabolism.

Direct evidence for HPr(Ser-P)/CcpA-mediatedregulation of the lac operon comes from studies with ccpAdisruption strains of S. thermophilus. Disruption of the ccpAgene impairs the growth of S. thermophilus on severalsugars as has been observed for other Gram-positivebacteria (Hueck et al., 1995; Egeter and Brückner, 1996;Monedero et al., 1997). The lacS promoter contains a cresite, overlapping the –10 box and the transcriptional startsite (Poolman, 1993), suggesting that the lacSZ expressionis repressed by CcpA (Henkin, 1996). In accordance,disruption of the ccpA gene in S. thermophilus CNRZ302results in derepression of lacSZ transcription duringexponential growth on lactose (van den Bogaard et al.,2000). Moreover, the rates of lactose uptake and galactoseexcretion are at least 4-fold increased in the ccpA disruptionstrain relative to wild-type cells. The increased lactoseuptake and hydrolysis does not result in an increased

growth rate on lactose, but leads to massive expulsion ofglucose into the fermentation medium. Apparently, loss ofa functional CcpA in S. thermophilus uncouples the controlof metabolism over transport and vice versa, as glycolysiscan no longer keep up with the massive lactose intake.Thus, the S. thermophilus ccpA disruption mutant has alactose transport capacity that exceeds the maximalglycolytic rate. The data indicate that a concerted activityof HPr(Ser-P) and CcpA results in fine-tuning of lactosetransport and hydrolysis capacity in order to accommodatemaximal glycolytic flux.

Although most S. thermophilus strains cannot usegalactose as a carbon source, galactose-fermentingmutants of strain CNRZ302 are readily obtained (Hutkinset al., 1985; Vaughan et al., 2001). Galactose is taken upby the LacS protein (Poolman, 1993), and fermented viathe Leloir pathway (Vaughan et al., 2001), but growth isslower than with lactose. When ccpA is disrupted in thegalactose-fermenting strain, derepression of lacSZtranscription is not observed during growth on galactose(van den Bogaard et al., 2000). This suggests that theglucose moiety derived from lactose induces repressionof the lacS promoter. The repression is not observed whenglucose is present in the growth medium, which is due tothe low rate of uptake of glucose. The LacS transportprotein of S. thermophilus, on the other hand, constitutesa very fast and efficient system for lactose uptake, leadingto high intake of glucose into glycolysis. This results inrelatively high intracellular HPr(Ser-P) concentrations(Deutscher et al., 1995) and, consequently, repression ofthe lacS promoter. Repression of the lac operon in S.thermophilus is not carbon-source dependent but isdetermined by the rate of glycolysis relative to sugar uptake.Current studies aim at relating glycolytic activity in S.thermophilus to catabolite control (van den Bogaard et al.,unpublished results). Analysis of the steady state pool ofglycolytic intermediates isolated from wild-type and ccpA-disruption strains grown on different carbon sources shouldelucidate the key-glycolytic steps controlled by HPr(Ser-P) and CcpA. Probably, FDP, PEP and/or ATP function asthe intracellular indicators of this glycolytic flux, as has beensuggested for other Gram-positive bacteria (Deutscher andEngelman 1984; Reizer et al., 1984; 1989b; 1998; Galinieret al., 1998; Kravanja et al., 1999 and Dossonnet et al.,2000; Jault et al., 2000).

Recently, the S. thermophilus ptsHI operon, encodingHPr and Enzyme I, has been cloned and several HPrmutants have been constructed that abolish or mimicphosphorylation at residues His15 or Ser46 (van denBogaard et al., unpublished results). The expression ofthese mutants in a ptsH knock-out strain will give moreinsight in the physiological role of the differentphosphorylated forms of HPr in the regulation of lactosemetabolism (Deutscher et al., 1994; Ye et al., 1994 a, b;Luesink et al., 1998). Furthermore, expression of HPrmutants that are affected in their affinity for IIALacS or theirphosphorylation capacity could yield further evidence forthe role of HPr in the modulation of the LacS exchangeactivity as indicated by the in vitro experiments.

Regulation of Carbohydrate Utilization 409

Model for Autoregulation of Lactose Transport and

Metabolism in S. thermophilus

On the basis of knowledge described in the previoussections a model has been proposed for the control oflactose transport and metabolism in S. thermophilus (Figure4). The rate of lactose transport via LacS is very susceptibleto the lactose/galactose ratio in the growth-medium as thetransporter has a higher affinity for galactose (end-productof the fermentation) than for the substrate lactose. Thisimplies that the transport capacity will decrease whengalactose accumulates in the medium even with millimolarconcentrations of lactose available. At some point duringgrowth this will be reflected in a reduced glycolytic activity,which affects the concentrations of different glycolyticintermediates to which the HPr(Ser-P)/HPr(His~P) ratio isvery sensitive. ATP is an effector of HPr(Ser) kinase,whereas Pi is an inhibitor. Besides, controllingHPr(Ser)kinase, the HPr(Ser-P)phosphatase is stimulatedby Pi and inhibited by ATP (Deutscher and Saier, 1983;Reizer et al., 1984; 1989b; Deutscher et al., 1985). Theintracellular concentrations of ATP, PEP and Pi vary inresponse to the carbohydrate availability as has been firmlyestablished for other lactic acid bacteria. ATP levels arerelatively high in rapidly metabolizing cells, whereas Pi andPEP are low under these conditions. These lattercompounds become high at the end of the exponentionalphase of growth and remain high in the stationary phase(Mason et al., 1981; Thompson and Torchia, 1984; Koningset al., 1989). These physiological parameters form the basisfor the proposed changes in enzyme activity when theglycolytic activity decreases. Increasing PEPconcentrations will induce a rise in the HPr(His~P)concentration due to PEP-dependent Enzyme I

phosphorylation of HPr. Consequently, the LacS proteinbecomes phosphorylated, which in turn increases themaximal transport rate of the LacS protein. At the sametime, the modified activities of HPr(Ser)kinase and HPr(Ser-P)phosphatase will decrease the HPr(Ser-P) concentration.Accordingly, a relief of the HPr(Ser-P)/CcpA-mediatedrepression of the lacS promoter will result in the synthesisof more LacS and β-galactosidase, which in turn will providemore glucose for glycolysis. In this way the uptake oflactose is tuned to the lactose/galactose ratio in the mediumas well as the glycolytic capacity of the cell. Thisautoregulatory mechanism allows S. thermophilus to co-metabolize lactose and sucrose.

Concluding Remarks

Hierarchical control of carbohydrate transport andmetabolism results in the preferential utilization of onecarbohydrate over another. Autoregulatory control, on theother hand, adjusts the activity of the first step(s) ofmetabolism, most often the transport activity, to themetabolic capacity of the cell and to the availability of aparticular substrate. In both cases, regulation of the activityof existing transporters/enzymes as well as gene regulationplays a role. For both regulatory processes, componentsof the PTS form the underlying signaling pathway thatsenses the availability of carbon sources and the metabolicneeds of the cell to respond to these signals. It does so viamodulation of the phosphorylation state of the PTScomponents in a PEP/Enzyme I or ATP/HPr(Ser)kinasedependent manner. Specific PTS components are theeffectors that control transport and metabolism of severalcarbohydrates. In Gram-negative enteric bacteria IIAGlc is

Figure 4. Schematic representation of the regulation of lactose transport capacity in S. thermophilus. The arrows in the boxes represent a decrease orincrease of the concentration or activity of the indicated component.

410 Gunnewijk et al.

the critical effector molecule (Figure 1), whereas in Gram-positive bacteria HPr assumes this role (Figure 2). Theregulation of transport and metabolism of the non-PTSsugar lactose in S. thermophilus is unprecedented andshows how metabolism is fine-tuned to the carbohydrateavailability and metabolic capacity of the cell (Figure 5).We can conclude that evolution has generated a wide rangeof regulatory mechanisms for the control of carbohydrateutilization, but in most, if not all, of these mechanisms, thecomponents of the PTS system form the intricate pathwayto sense the changes and convert the signal(s) into anadequate response.

Acknowledgements

This work has been supported by grants from the Dutch Foundation forLife Sciences (ALW) and the EU framework 4 Program (BIO-4CT-960439).

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Figure 5. Regulation of lactose transport in S. thermophilus. Schematic representation of HPr(His~P)-mediated phosphorylation of LacS (stimulation oflactose transport activity), and HPr(Ser-P)/CcpA-mediated regulation of lacS transcription. The depicted symbols are described in the legends of Figure 2.

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RNA polym. CcpA

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